An RNAi System in Physcomitrella patens with an Internal Marker for Silencing Allows for Rapid Identification of Loss of Function Phenotypes

• DOI: 10.1055/s-2005-837597
• Publication Date: 2005-05-23T13:19:52Z
• Authors: M. Bezanilla; P.‐F. Perroud; A. Pan; P. Klueh; R. S. Quatrano
• Keywords: Physcomitrella patens; Reverse Genetics; Loss function; Cloning (programming)

Abstract: RNAi is a powerful method for generating loss of function mutants, especially targeting genes belonging to large gene families. We have recently shown that functions in the moss Physcomitrella patens. obtained stable lines show constitutive silencing nuclearly localized GFP:GUS fusion protein (NLS:GFP:GUS). However, display do not necessarily reduced transcript levels. Therefore, system has been developed silences NLS:GFP:GUS reporter construct at same time as it interest. Gateway™ (Invitrogen) recombination cassettes were incorporated into these vectors facilitate cloning many different cDNA sequences. In addition, generated contain genomic DNA sequence information increase production lines. Transformation with constructs results strong within 24 h and least month after transformation. FtsZ2‐1, whose phenotype known, was test case analyzing phenotypes. One hundred per cent regenerating colonies silenced GFP exhibit FtsZ2‐1 phenotype, validating use this assay phenotypes plant unknown function.